The present invention relates to a recombinant organism that expresses heterologous DNA coding for the elements of an alternative pathway for glucose uptake, in particular, a pathway utilized in nature by Zymomonas mobilis.
The cost of microbial production of chemicals is typically dominated by the cost of the sugar feedstock. Glucose is a basic starting point for the microbial production of commercial products such as amino acids, vitamins, citrate, succinate, fumarate, citrate, lactic acid, acetic acid, ethanol and solvents. Processes for production of various chemicals from glucose strive to maximize the efficiency with which the carbon skeleton of glucose is converted into the desired product.
Phosphoenol pyruvate (PEP) is a central intermediate in glucose metabolism, residing at a branch point for the biosynthesis of many compounds of commercial importance. For example, an equimolar amount PEP is combined with erythrose-4-phosphate to provide the carbon skeleton for aromatic products such as tyrosine, phenylalanine, tryptophan, and some vitamins among other compounds. PEP also is combined with carbon dioxide to produce oxaloacetic acid in the Tricarboxylic Acid (TCA) Cycle. Oxaloacetic acid serves as the carbon skeleton for aspartic acid, asparagine, threonine, isoleucine, methionine, diaminopimelic acid, lysine, pyrimidines, and a host of intermediates. By a reverse of the TCA cycle, oxaloacetic acid is converted to fumarate and succinate. Succinate serves as the carbon backbone for tetrapyrrole biosynthesis. In the forward TCA cycle, oxaloacetic acid is combined with acetyl-CoA to form citric acid, .alpha.-ketoglutarate and succinate among other intermediates. .alpha.-Ketoglutarate serves as the primary carbon skeleton for the synthesis of glutamic acid, ornithine, arginine, citrulline, polyamines, and glutamine and many intermediates. Thus, the conversion of PEP to oxaloacetic acid serves as a primary route to supply TCA Cycle intermediates which can be used for biosynthesis. In many bacteria, PEP also is necessary for the transport of glucose into the cell. Glucose is phosphorylated in a concerted process by a multiprotein, -membrane-bound complex termed the phosphotransferase system (PTS). In this process, PEP serves as the source of a high energy phosphate which is ultimately attached to glucose to yield glucose-6-phosphate and pyruvate. During glycolysis in these organisms, half of the PEP produced is obligately consumed to provide energy for glucose uptake. This reduces by 50% the amount of PEP available as a source of carbon skeletons for biosynthesis, severely impacting the efficiency of conversion into many desired commercial products.
The ethanol-producing bacterium Z. mobilis has an alternative mechanism for glucose uptake that utilizes ATP rather than PEP as an energy source. In this organism, glucose selectively enters via a membrane permease, designated "glucose-facilitated diffusion protein" (GLF), which has a deduced size of approximately 50 kilodaltons (kDa). The intracellular glucose subsequently is phosphorylated by a glucose-specific hexokinase called glucokinase (GLK) using ATP to produce glucose-6-phosphate and ADP. This glucose-6-phosphate is chemically identical to the glucose-6-phosphate produced by the PTS pathway. A similar alternative pathway exists in yeast, although the hexokinase of yeast is not glucose-specific.
Although Z. mobilis has simple nutritional requirements, the range of sugars metabolized by this organism is very limited and normally consists of glucose, fructose and sucrose. Zymomonas mobilis is incapable of growth without a fermentable sugar, even in rich medium such as nutrient broth. Substrate-level phosphorylation from the fermentation of these simple sugars is the sole source of energy for biosynthesis and homeostasis. That is, Z. mobilis is an obligately fermentative bacterium which lacks a functional system for oxidative phosphorylation. In the absence of a functioning electron transport system for oxidative phosphorylation, a large proportion of the PEP produced is consumed in short pathways which regenerate NAD.sup.+, an obligate requirement for continued glycolysis and ATP production. In addition to being an obligatively fermentative bacterium that is limited with respect to the variety of feedstocks that it can metabolize, Z. mobilis also tends to be a relatively demanding organism in terms of culture conditions.
Recently, Barnell et al. cloned and sequenced the Z. mobilis GLKgene (glk) and showed it to be expressed in an active form in E. coli, J. Bacteriol. 172:7227 (1990). A 1,422 base pair open reading frame, characterized by partial homology with both the xylose permease gene in E. coli and a human glucose transporter, also was discovered, approximately 3 kilobase pairs upstream from the glk gene. It was hypothesized that this open reading frame corresponded to the structural sequence of the Z. mobilis GLF gene (glf). Production of GLF in recombinant E. coli was not observed by Barnell et al., however, and a plasmid containing both the glk and glf genes was not constructed.
Prior to the present invention, it would have been doubtful whether the GLF gene would express functional protein in an organism other than Z. mobilis. Unlike soluble cytoplasmic enzymes that have been readily expressed as functional proteins in E. coli, a membrane permease protein must be correctly inserted into the cytoplasmic membrane and form integral interactions with the membrane lipids to ensure function. The expression of functional membrane proteins in heterologous systems is problematic. Although function occurs in some cases, heterologous membrane proteins often fail to function in E. coli. Failure to function is presumed to be due in part to differences between signal sequences for membrane insertion and differences in membrane lipid compositions. Barnell et al. do not suggest or provide any evidence that the glf-encoded protein was functional in E. coli.
Zymomonas mobilis has a unique membrane lipid composition. It has been hypothesized that this composition represents an evolutionary adaptation that allows Z. mobilis to survive in the presence of high levels of ethanol. This membrane composition in Z. mobilis is very different from that of other microorganisms, including microbes like E. coli and other enteric bacteria, a factor militating against insertion of membrane proteins into E. coli. For example, Z. mobilis contains different phospholipids than E. coli. Positively charged phosphatidyl choline is abundant in Z. mobilis, but totally absent in E. coli. Phosphatidyl choline is synthesized from phosphatidyl ethanolamine, a conversion that forms the basis for many metabolic control systems in eukaryotic organisms.
The membrane phospholipids of Z. mobilis also differ with respect to the fatty acid composition. In Z. mobilis, the fatty acid composition is almost exclusively 18:1 vaccenic acid. In contrast, E. coli phospholipids contain a mixture of shorter chain saturated and unsaturated fatty acids which change in response to growth temperature as a part of the temperature adaptation process.
An additional feature unique to Z. mobilis is the presence of abundant hopanoids. Hopanoids either are present at very low levels or are absent in E. coli. Hopanoids have been proposed to serve as steroid equivalents in prokaryotic membranes and are hypothesized to be important for ethanol tolerance in Z. mobilis. Another unique class of membrane lipids in Z. mobilis is the ceramides. Ceramides are common in eukaryotes but are very unusual in bacteria. Thus, many differences exist in membrane compositions of Z. mobilis and E. coli which may be related to their respective ranges of environmental tolerance.
Molecular genetics offers the potential to combine in a single organism the ability to metabolize efficiently the entire range of biomass-derived sugars with the ability to use a pathway for glucose uptake that is not obligately coupled to PEP. For example, by modifying an enteric bacteria such as E. coli to use an alternative pathway for glucose uptake characteristic of Z. mobilis, the output of any synthetic product derived from a PEP precursor could be doubled because glucose transport into the cells would not be obligately coupled to PEP. As discussed above, there have been reasons to doubt heretofore that a recombinant organism containing the glf gene would exhibit the alternative pathway for glucose transport because of the significant differences between the membrane lipid composition of Z. mobilis and other organisms.